A commonly encountered situation in genetic analysis entails the need to identify a low percent of variant DNA sequences (‘target sequences’) in the presence of a large excess of non-variant sequences (‘reference sequences’). Examples for such situations include: (a) identification and sequencing of a few mutated alleles in the presence of a large excess of normal alleles; (b) identification of a few methylated alleles in the presence of a large excess of unmethylated alleles (or: vice versa) in epigenetic analysis; (c) identification and genotyping of a few fetal DNA sequences circulating in the blood of the mother where a large excess of mother's DNA sequences are also present; and (d) identification of tumor-circulating DNA in blood of cancer patients (or people suspected of having cancer) in the presence of a large excess of wild-type alleles.
While reliable high throughput screening methods for germline or high-prevalence somatic mutations have recently been described (Thomas, R. K., et al. (2007) Nat Genet, 39, 347-351; Chou, L. S., et al. (2005) Am J Clin Pathol, 124; 330-338; Thomas, R. K., et al. (2006) Nat Med, 12; 852-855) detection of low-prevalence somatic mutations in tumors with heterogeneity, stromal contamination or in bodily fluids is still problematic. And yet, the clinical significance of identifying these mutations is major in several situations. For example: (a) in lung adenocarcinoma, low-level EGFR mutations that cannot be identified by regular sequencing can confer positive response to tyrosine kinase inhibitors (Paez, J. G., et al. (2004) Science, 304; 1497-1500.) or drug resistance (Janne, P. A., et al. (2006) Clin Cancer Res, 12; 751-758) (b) mutations in plasma useful as biomarkers for early detection (Diehl, F., et al. (2005) Proc Natl Acad Sci USA, 102; 16368-16373) or tumor response to treatment (Kimura, T., et al. (2004) Ann N Y Acad Sci, 1022; 55-60) cannot be sequenced using conventional methods; and (c) mutations in tumors with frequent stromal contamination, such as pancreatic or prostate, can be ‘masked’ by presence of wild-type alleles, thus requiring laborious micro-dissection or missing mutations altogether.